Effects on Dry Matter Accumulation and Nitrogen

The interaction between the ATP-dependent evolution of H2 catalyzed by nitrogenase and the oxidation of H2 via a hydrogenase has been postulated to influence the efficiency of the N2-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna uguiculats L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H2 in air and the H2 produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N2 fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H2 in air. A comparison ofAnoka soybeans inoculated with the two different strains of R.japonicum showed that mean rates of C2H2 reduction and 02 consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different. When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N2 fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H2-evolving strain 176A27 produced increases in plant dry weight and total N2 fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N2 fixation for nodulated legumes capable of reutilizing the H2 evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H2-recycling process in N2-flxing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase. utilization during N2 fixation and therefore increase the quantity of N2 fixed and plant yield. The function of hydrogenase within legume nodules has been discussed by Dixon (6, 7) who has proposed the following possible roles: (a) serves as a means for reducing H2 concentrations in nodules below inhibitory levels; (b) acts as a mechanism for protecting nitrogenase from 02 inactivation by using H2 as a substrate to support respiration; and (c) provides a system whereby the H2 evolved from nitrogenase is metabolized and a portion of the otherwise wasted energy is conserved (4, 6). The proposal that H2 metabolism leads to ATP synthesis or to available reducing power which increases the efficiency of energy utilization within the nodule has been favoted in most discussions (1, 6-8, 15). Dixon (6) has shown that the oxidation of H2 via hydrogenase in pea nodule bacteroids apparently is linked to ATP production. At this time there are no published reports supporting the conclusion that hydrogenase in nodules is capable of providing the reducing potential via the appropriate electron carriers, to support N2 fixation directly. If energy is the major limitation of N2 fixation in leguminous species as has been proposed (4, 9), then the coupling of the oxidation of H2 to energy-yielding processes conceivably could increase the rate of N2 fixation. Alternatively the conservation of energy through H2 recycling processes might decrease the demand for photosynthate and consequently contribute to increased dry matter. production. The possibility also must be considered that oxidation of H2 via hydrogenase is not coupled to useful energy-producing processes and therefore is not beneficial to the plant. This paper describes an investigation for the purpose of comparing the yield and efficiency of N2 fixation by legumes with nodules that recycle H2, with those that lack an active hydrogenase and evolve the H2 produced during N2 fixation. In regard to their capability to catalyze nitrogenase-dependent H2 evolution and H2 oxidation via a hydrogenase, leguminous symbionts that have been tested may be classified into two categories (13, 15). The first includes symbionts that evolve H2 produced by the nitrogenase system. The second group is like the first, with the exception that H2 produced via the nitrogenase system is recycled via a hydrogenase. So far it appears that all in vivo N2-fixing systems reduce protons during the N2-fixing process. Our investigations have concentrated on testing the premise that the capacity of nodules to recycle the H2 from the nitrogenase system would be expected to improve the efficiency of energy ' This research was funded by grants to H. J. E. from the National Science Foundation (PCM 74-17812-A02), The Rockefeller Foundation (GA AS-7628), and by the Oregon Agricultural Experimental Station (Technical Paper No. 4570). 2 Present address: Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824. 398 MATERIALS AND METHODS Seeds were surface-sterilized by immersion for I min in 95% ethyl alcohol and then 5 min in 0.2% acidified HgCl2 (17). Residual HgCl2 was removed by rinsing 10 times with sterile distilled H20. Seeds were placed on sterile water agar plates in the dark at 26 C where they germinated. After 2 days soybean seedlings (Glycine max L. Merr cv. Anoka) were selected and inoculated with Rhizobiumjaponicum strains USDA I 10 or USDA 31 and cowpea seedlings (Vigna unguiculata L. Walp. cv. Whippoorwill) with Rhizobium strains 176A28 or 176A27 and transplanted into surface-sterilized 20-cm plastic pots filled with Perlite. The pot cultures with seedlings were kept moist for 3 days and then irrigated with N-free nutrient solution as described previously (13). Plants were grown in the greenhouse with supplemental fluorescent lighting of about 5380 lux at 0.73 meter on a 16-hr photoperiod. The day temperature was maintained near 29 C, the night temperature near 24 C. Ten replicate pot cultures each containing four soybean plants or five cowpea plants were used for each strain at each harvest date. Experiments were arranged in a completely randomized design in the greenhouse. www.plantphysiol.org on July 13, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. HYDROGEN REACTIONS OF LEGUMES. II Rates of C2H2 reduction, H2 evolution, and O2 utilization of excised nodules were performed as described previously (14) except rates of C2H2 reduction were measured on separate samples of nodules. Gases were obtained from the same sources as listed previously (14). At least 2 days prior to each experiment, plants in the pot cultures were transferred into a growth chamber (day temperature, 29 C; night temperature, 24 C; light intensity, 22,000 lux and photoperiod, 16 hr). Samples of nodules with attached root pieces (about 0.3 g) were excised and used for C2H2 reduction and for H2 evolution and 02 uptake measurements. The remaining nodules were removed and weighed. The entire plants including nodules were dried at 70 C, weighed, and ground. After Kjeldahl digestion of 0.2-g samples, nitrogen contents were determined on diluted aliquots of the digested material by use of an ammoniasensitive electrode (Orion 95-10-00) as described by Bremner (3). Analyses of variance were calculated for the data presented with the exception of relative efficiencies.